U.S. patent number 9,080,183 [Application Number 12/664,399] was granted by the patent office on 2015-07-14 for promoter.
This patent grant is currently assigned to HOFFMANN-LA ROCHE INC.. The grantee listed for this patent is Christian Klein, Erhard Kopetzki. Invention is credited to Christian Klein, Erhard Kopetzki.
United States Patent |
9,080,183 |
Klein , et al. |
July 14, 2015 |
Promoter
Abstract
The current invention reports a promoter having the nucleic acid
sequence of SEQ ID NO: 02, or SEQ ID NO: 03, or SEQ ID NO: 04, or
SEQ ID NO: 06, which is a 5' shortened SV40 promoter with reduced
promoter strength especially useful for the limited expression of
heterologous polypeptides or selectable markers.
Inventors: |
Klein; Christian (Iffeldorf,
DE), Kopetzki; Erhard (Penzberg, DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
Klein; Christian
Kopetzki; Erhard |
Iffeldorf
Penzberg |
N/A
N/A |
DE
DE |
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Assignee: |
HOFFMANN-LA ROCHE INC. (Nutley,
NJ)
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Family
ID: |
38476213 |
Appl.
No.: |
12/664,399 |
Filed: |
June 25, 2008 |
PCT
Filed: |
June 25, 2008 |
PCT No.: |
PCT/EP2008/005135 |
371(c)(1),(2),(4) Date: |
December 14, 2009 |
PCT
Pub. No.: |
WO2009/003622 |
PCT
Pub. Date: |
January 08, 2009 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20100159489 A1 |
Jun 24, 2010 |
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Foreign Application Priority Data
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Jun 29, 2007 [EP] |
|
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07012772 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
14/4702 (20130101); C12N 15/85 (20130101); C12N
15/63 (20130101); C07K 14/43595 (20130101) |
Current International
Class: |
C12N
15/00 (20060101); C12N 15/85 (20060101); C07K
14/47 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2006-050956 |
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Feb 2006 |
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JP |
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WO 9962927 |
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Dec 1999 |
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WO |
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Other References
Hartzell, S.W. et al., Proc. Natl. Acad. Sci 81:23-27 ( 1984).
cited by applicant .
(Translation of Korean Office Action 2009-70270412011). cited by
applicant .
(NCBI, GenBank, Accession No. EF550208.1 May 23,2007). cited by
applicant .
(Translation of Jap Off Act in Corres Jap Appl 2010513746 May 10,
2012). cited by applicant .
Osamu Kanemitsu, Introduction to Antibody Engineering (Chijin
Shokan Co., Ltd.),:198-201 (Jan. 25, 1994). cited by applicant
.
(Translation of Russian Off Act in Corres Appl 2010102812 Feb. 2,
2012). cited by applicant .
Fromm M. et al: Journal of Molecular and Applied Genetics, vol.
1:5, (1982), pp. 457-481, XP008083910. cited by applicant .
Fromm M. et al: Journal of Molecular and Applied Genetics, vol.
2:1, (1983), pp. 127-135, XP008083908. cited by applicant .
Gorman C et al, Molecular and Cellular Biology, 2:9 (1982) 1044-105
XP000574017. cited by applicant .
Dynan W et al, Cll, 35:1 (1983) 79-88 XP002451926. cited by
applicant .
Benoist C et al, Nature, 290:5804 (1981) 304-310 XP008083948. cited
by applicant .
Rio D et al, Journal of Molecular and Applied Genetics, 2:5 (1984)
423-435 XP008083924. cited by applicant .
Everett R et al, Nucleic Acids Research 11:8 (1983) 2447-2464.
cited by applicant .
Firak T et al Molecular and Cellular Biology, 6:11, (1986)
3667-3676 XP002495690. cited by applicant .
Gong, S et al, Virology 163:2 (1988) 481-493. cited by
applicant.
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Primary Examiner: Qian; Celine
Claims
The invention claimed is:
1. A method for the selection of a cell expressing a heterologous
polypeptide to the cell expressing it comprising the following
steps: a) transfecting an isolated eukaryotic cell with a nucleic
acid comprising i) a first expression cassette comprising a nucleic
acid encoding a heterologous polypeptide, ii) a second expression
cassette comprising a first nucleic acid comprising the sequence of
SEQ ID NO: 04 and a second nucleic acid encoding a selectable
marker selected from the group consisting of hygromycin
phosphotransferase, neomycin and G418 aminoglycoside
phosphotransferase, dLNGFR and GFP, whereby said first and second
nucleic acid are operably linked, b) cultivating said transfected
cell under conditions suitable for the growth of non-transfected
cell; and c) cultivating said cells under selective culture
conditions; d) selecting a cell propagating in step b) and under
selective culture conditions in step c).
2. The method of claim 1, wherein step d) of said method is
selecting a cell propagating in step b) and expressing the
selectable marker encoded by said second nucleic acid.
3. A method for the expression of a heterologous polypeptide to the
cell expressing it, comprising the following steps: a) transfecting
an isolated eukaryotic cell with a nucleic acid comprising an
expression cassette comprising a first nucleic acid having the
sequence of SEQ ID NO: 04 operably linked to a second nucleic acid
encoding a heterologous polypeptide, b) selecting a cell
transfected in step a), c) cultivating the selected cell of step b)
under conditions suitable for the expression of said heterologous
polypeptide; and d) recovering the heterologous polypeptide from
the cell or the cultivation medium.
4. The method of claim 3, wherein the nucleic acid comprises a
second expression cassette encoding an aminoglycoside
phosphotransferase selected from the group consisting of hygromycin
phosphotransferase, neomycin and G418 aminoglycoside
phosphotransferase.
5. The method of claim 1, wherein said eukaryotic cell is a
mammalian cell.
6. The method of claim 5, wherein said mammalian cell is a CHO
cell, a BHK cell, a HEK cell or, a Sp2/0 cell.
7. The method of claim 6, wherein said mammalian cell is a CHO cell
or a HEK cell.
8. The method of claim 1, wherein said heterologous polypeptide is
an immunoglobulin, or an immunoglobulin-fragment, or an
immunoglobulin-conjugate.
9. The method of claim 3, wherein said first nucleic acid is a
nucleic acid having the sequence of SEQ ID NO: 04 and has a
promoter strength of 20% or less of the SV40 promoter of SEQ ID NO:
05 when operably linked to the nucleic acid of SEQ ID NO: 07.
Description
This application is the National Stage of International Application
No. PCT/EP2008/005135 filed Jun. 25, 2008, which claims the benefit
of EP 07012772.5 filed Jun. 29, 2007, which is hereby incorporated
by reference in its entirety.
The current invention is in the field of protein expression and
cell selection. It is herein reported a promoter with low promoter
strength and thus with a limited expression of an operably linked
coding nucleic acid.
BACKGROUND OF THE INVENTION
The expression of proteins is a fundamental process in living
cells. All information required for protein expression is provided
by a single nucleic acid. This nucleic acid not only contains the
information of the protein's amino acid sequence, it also provides
the regulatory information required (e.g. the ribosomal binding
site, the start and end-signals for transcription, splice signals,
enhancer elements, etc.) including a promoter/promoter
sequence.
A promoter is a nucleic acid that regulates the amount of
transcription of a nucleic acid, e.g. encoding a polypeptide, to
which it is operably linked, into pre-mRNA. It is a transcription
control element, which is located around the RNA polymerase
initiation site at the 5'-end of an operably linked coding
sequence. From analysis of the SV40 early promoter it is known that
recognition/binding sites for transcription activators are
contained in promoters in segments consisting of 7-20 basepairs.
One segment is the start site for RNA synthesis, e.g. the well
known TATA-box. Other segments, located approximately 30-110
basepairs 5', i.e. upstream, to the start site for RNA synthesis,
are defining the frequency of transcription initiation. A promoter
at least requires one segment that initiates RNA synthesis at a
specific site and in a defined direction, i.e. in 5' to 3'
direction.
Known promoters are the lac-lpp, the ara-, the lac-, the tac-, the
trc-, the trp-, the phoA-, the P.sub.BAD-, the .lamda..sub.PL-, the
lpp-, and the T7-promoter. The SV40 promoter is a nucleic acid
sequence derived from the genome of Simian (vacuolating) Virus 40.
For the recombinant production of a heterologous polypeptide in a
eukaryotic or prokaryotic cell normally one or more expression
plasmids are introduced into the cell. The expression plasmid(s)
comprises an expression cassette for the expression of a
heterologous polypeptide and also an expression cassette for the
expression of a selectable marker, which is required for the
selection of transfected cells expressing the heterologous
polypeptide. The synthesis of the heterologous polypeptide and of
the selectable marker both requires a fraction of the cell's
expression machinery's capacity.
As it is the aim to produce predominantly the heterologous
polypeptide most of the available capacity of the cell's expression
machinery should be allocated to the expression of the nucleic acid
encoding the heterologous polypeptide. Only a minor amount should
be used for the expression of the selectable marker. This
allocation of expression capacity is done via the strength of the
corresponding promoters. The stronger a promoter is the more of the
operably linked nucleic acid is transcribed and thus translated.
Therefore, it exists a need for promoters with adjustable or
reducible promoter strength.
Taylor, W. E., et al. (Endocrinol. 137 (1996) 5407-5414) report
human stem cell factor promoter deletion variants. In US patent
application US 2007/0092968 novel hTMC promoter and vectors for the
tumor-selective and high-efficient expression of cancer therapeutic
genes is reported. Fromm et al. (J. Mol. Appl. Gen. 1 (1982)
457-481 and ibid 2 (1983) 127-135) report deletion mapping and
deletion mutants of SV-40 early region promoter. Chitinase
chitin-binding fragments are reported in U.S. Pat. No. 6,399,571.
WO 99/62927 reports connective tissue growth factor-4.
SUMMARY OF THE INVENTION
The first aspect of the current invention is a promoter having,
i.e. with, a nucleic acid sequence of SEQ ID NO: 02 or SEQ ID NO:
03 or SEQ ID NO: 04 or SEQ ID NO: 06. In one embodiment the
promoter has the nucleic acid sequence of SEQ ID NO: 04.
A second aspect of the current invention is a nucleic acid that has
the nucleotide sequence of SEQ ID NO: 04 and that has a promoter
strength of 20% or less compared to the wild-type SV40 promoter of
SEQ ID NO: 05 when operably linked to the nucleic acid sequence of
SEQ ID NO: 07 encoding the green-fluorescent-protein (GFP).
A further aspect of the current invention is a method for the
selection of a cell comprising the following steps in this order:
a) transfecting a eukaryotic cell with a nucleic acid comprising i)
a first expression cassette comprising a nucleic acid encoding a
heterologous polypeptide, ii) a second expression cassette
comprising a first nucleic acid of SEQ ID NO: 04 and a second
nucleic acid encoding a selectable marker, whereby the first
nucleic acid is operably linked to the second nucleic acid, b)
cultivating said transfected cell under conditions suitable for
growth of the non-transfected eukaryotic cell, c) selecting a cell
propagating in step b) and also i) propagating under selective
culture conditions, or ii) expressing the selectable marker.
In one embodiment of this aspect of the invention the eukaryotic
cell is a mammalian cell. In a preferred embodiment the mammalian
cell is a CHO cell, BHK cell, or PER.C6.RTM. cell, or HEK cell, or
Sp2/0 cell. In another embodiment the heterologous polypeptide is
an immunoglobulin, or an immunoglobulin-fragment, or an
immunoglobulin-conjugate. In one embodiment the selectable marker
is a neomycin-aminoglycoside phosphotransferase, or a
hygromycin-phosphotransferase, or dLNGFR, or GFP.
A forth aspect of the current invention is a method for the
expression of a heterologous polypeptide which comprises the
following steps in this order: a) transfecting a mammalian cell
with a nucleic acid comprising an expression cassette comprising a
first nucleic acid of SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO:
04 or SEQ ID NO: 06 operably linked to a second nucleic acid
encoding a heterologous polypeptide, b) selecting a cell
transfected in step a), c) cultivating the selected cell under
conditions suitable for the expression of the heterologous
polypeptide, d) recovering the heterologous polypeptide from the
cell or the cultivation medium.
In one embodiment of this aspect of the current invention the
mammalian cell is a CHO cell, a BHK cell, or a PER.C6.RTM. cell, or
HEK cell, or Sp2/0 cell. In another embodiment the first nucleic
acid is of SEQ ID NO: 04. In a further embodiment the second
nucleic acid is encoding an immunoglobulin, or an
immunoglobulin-fragment, or an immunoglobulin-conjugate. In still
another embodiment the nucleic acid comprises a second expression
cassette encoding a selectable marker.
DETAILED DESCRIPTION OF THE INVENTION
The current invention reports a novel promoter nucleic acid with a
nucleotide sequence of SEQ ID NO: 02, or SEQ ID NO: 03, or SEQ ID
NO: 04, or SEQ ID NO: 06.
Methods and techniques useful for carrying out the current
invention are known to a person skilled in the art and are
described e.g. in Ausubel, F. M., ed., Current Protocols in
Molecular Biology, Volumes I to III (1997), and Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). As known
to a person skilled in the art enables the use of recombinant DNA
technology the production of numerous derivatives of a nucleic acid
and/or polypeptide. Such derivatives can, for example, be modified
in one individual or several positions by substitution, alteration,
exchange, deletion, or insertion. The modification or
derivatisation can, for example, be carried out by means of site
directed mutagenesis. Such modifications can easily be carried out
by a person skilled in the art (see e.g. Sambrook, J., et al.,
Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor
Laboratory Press, New York, USA). The use of recombinant technology
enables a person skilled in the art to transform various host cells
with heterologous nucleic acid(s).
A "promoter" refers to a nucleic acid, i.e. polynucleotide
sequence, which controls transcription of a nucleic acid to which
it is operably linked. A promoter may include signals for RNA
polymerase binding and transcription initiation. The promoter(s)
used will be functionable in the cell type of the host cell in
which expression of the operably linked nucleic acid is
contemplated. A large number of promoters including constitutive,
inducible, and repressible promoters from a variety of different
sources are well known in the art (and identified in databases such
as GenBank). They are available as or within cloned polynucleotides
(from, e.g., depositories such as ATCC as well as other commercial
or individual sources). A "promoter" comprises a nucleotide
sequence that directs the transcription of e.g. an operably linked
structural gene. Typically, a promoter is located in the 5'
non-coding or 5'-untranslated region (5'UTR) of a gene, proximal to
the transcriptional start site of a structural gene. Sequence
elements within promoters that function in the initiation of
transcription are often characterized by consensus nucleotide
sequences. These sequence elements include RNA polymerase binding
sites, TATA sequences, CAAT sequences, differentiation-specific
elements (DSEs; McGehee, R. E., et al., Mol. Endocrinol. 7 (1993)
551), cyclic AMP response elements (CREs), serum response elements
(SREs; Treisman, R., Seminars in Cancer Biol. 1 (1990) 47),
glucocorticoid response elements (GREs), and binding sites for
other transcription factors, such as CRE/ATF (O'Reilly, M. A., et
al., J. Biol. Chem. 267 (1992) 19938), AP2 (Ye, J., et al., J.
Biol. Chem. 269 (1994) 25728), SP1, cAMP response element binding
protein (CREB; Loeken, M. R., Gene Expr. 3 (1993) 253-264) and
octamer factors (see, in general, Watson et al., eds., Molecular
Biology of the Gene, 4th ed., The Benjamin/Cummings Publishing
Company, Inc. 1987, and Lemaigre, F. P. and Rousseau, G. G.,
Biochem. J. 303 (1994) 1-14). If a promoter is an inducible
promoter, then the rate of transcription increases in response to
an inducing agent. In contrast, the rate of transcription is not
regulated by an inducing agent if the promoter is a constitutive
promoter. Repressible promoters are also known. For example, the
c-fos promoter is specifically activated upon binding of growth
hormone to its receptor on the cell surface. Tetracycline (tet)
regulated expression can be achieved by artificial hybrid promoters
that consist e.g. of a CMV promoter followed by two Tet-operator
sites. The Tet-repressor binds to the two Tet-operator sites and
blocks transcription. Upon addition of the inducer tetracycline,
the Tet-repressor is released from the Tet-operator sites and
transcription proceeds (Gossen, M. and Bujard, H., Proc. Natl.
Acad. Sci. USA 89 (1992) 5547-5551). For other inducible promoters
including metallothionein and heat shock promoters, see, e.g.,
Sambrook, et al. (supra), and Gossen, M., et al., Curr. Opin.
Biotech. 5 (1994) 516-520. Among the eukaryotic promoters that have
been identified as strong promoters for high-level expression are
the SV40 early promoter, adenovirus major late promoter, mouse
metallothionein-I promoter, Rous sarcoma virus long terminal
repeat, Chinese hamster elongation factor 1 alpha (CHEF-1, see e.g.
U.S. Pat. No. 5,888,809), human EF-1 alpha, ubiquitin, and human
cytomegalovirus immediate early promoter (CMV IE). An enhancer
(i.e., a cis-acting DNA element that acts on a promoter to increase
transcription) may be necessary to function in conjunction with the
promoter to increase the level of expression obtained with a
promoter alone, and may be included as a transcriptional regulatory
element. Often, the polynucleotide segment containing the promoter
will include enhancer sequences as well (e.g., CMV or SV40).
The term "nucleic acid" as used herein, is a polymer consisting of
individual nucleotides, i.e. a polynucleotide. It refers to a
naturally occurring, or partially or fully non-naturally occurring
nucleic acid, which is e.g. encoding a polypeptide that can be
produced recombinantly. The nucleic acid can be build up of
DNA-fragments which are either isolated or synthesized by chemical
means. The nucleic acid can be integrated into another nucleic
acid, e.g. in an expression plasmid or the genome/chromosome of a
host cell. Plasmid includes shuttle and expression vectors.
Typically, the plasmid will also comprise a prokaryotic propagation
unit comprising an origin of replication (e.g. the ColE1 origin of
replication) and a selectable marker (e.g. ampicillin or
tetracycline resistance gene) for replication and selection,
respectively, of the vector in bacteria.
The term "promoter strength" and grammatical equivalents thereof as
used within the current invention denotes the efficacy of a
promoter in the transcription of an operably linked nucleic acid.
The promoter strength of a promoter can be high, i.e. it can be of
from 90% to more than 100%, or medium, i.e. it can be of from 40%
to less than 90%, or low, i.e. it can be up to less than 40%, if
compared to the promoter strength of the wild-type SV40 promoter of
SEQ ID NO: 05. This value can be determined by comparing the amount
of expression of a heterologous polypeptide operably linked to the
promoter in question to the amount of expression of the
heterologous polypeptide operably linked to the wild-type SV40
promoter in the same cell type. This can be done e.g. by
determining the amount of expression of the heterologous
polypeptide in a CHO- or HEK-cell transfected with an expression
cassette consisting of the promoter in question operably linked to
a nucleic acid encoding the heterologous polypeptide by an
ELISA-assay. By comparing this amount to the amount of expression
of the same heterologous polypeptide in the same cell line
transfected with an expression cassette consisting of the wild-type
SV40 promoter operably linked to a nucleic acid encoding the
heterologous polypeptide determined with the same ELISA-assay i.e.
comparing the amount of heterologous polypeptide in the same cell
with the same expression plasmid wherein only the promoter is
changed, the relative promoter strength can be determined. The term
"wild-type SV40 promoter" as used within this application denotes a
nucleic acid of SEQ ID NO: 05 which correspond to position 72-411
of the nucleic acid of SEQ ID NO: 01, which is the genome of the
SV40.
"Operably linked" refers to a juxtaposition of two or more
components, wherein the components so described are in a
relationship permitting them to function in their intended manner.
For example, a promoter and/or enhancer are operably linked to a
coding sequence, if it acts in cis to control or modulate the
transcription of the linked coding sequence. Generally, but not
necessarily, the DNA sequences that are "operably linked" are
contiguous and, where necessary to join two protein encoding
regions such as a secretory leader/signal sequence and a
polypeptide, contiguous and in reading frame. However, although an
operably linked promoter is generally located upstream of the
coding sequence, it is not necessarily contiguous with it.
Enhancers do not have to be contiguous. An enhancer is operably
linked to a coding sequence if the enhancer increases transcription
of the coding sequence. Operably linked enhancers can be located
upstream, within, or downstream of coding sequences, and at
considerable distance from the promoter. A polyadenylation site is
operably linked to a coding sequence if it is located at the
downstream end of the coding sequence in such a way that
transcription proceeds through the coding sequence into the
polyadenylation sequence. Linking is accomplished by recombinant
methods known in the art, e.g., using PCR methodology, and/or by
ligation at convenient restriction sites. If convenient restriction
sites do not exist, then synthetic oligonucleotide adaptors or
linkers are used in accord with conventional practice.
Within the scope of the present invention, transfected cells may be
obtained with substantially any kind of transfection method known
in the art. For example, the nucleic acid may be introduced into
the cells by means of electroporation or microinjection.
Alternatively, lipofection reagents such as FuGENE 6 (Roche
Diagnostics GmbH, Germany), X-tremeGENE (Roche Diagnostics GmbH,
Germany), and LipofectAmine (Invitrogen Corp., USA) may be used.
Still alternatively, the nucleic acid may be introduced into the
cell by appropriate viral vector systems based on retroviruses,
lentiviruses, adenoviruses, or adeno-associated viruses (Singer,
O., Proc. Natl. Acad. Sci. USA 101 (2004) 5313-5314).
The term "cell" or "host cell" refers to a cell into which a
nucleic acid, e.g. encoding a heterologous polypeptide or
constituting an shRNA, can be or is introduced/transfected. Host
cells include both prokaryotic cells, which are used for
propagation of vectors/plasmids, and eukaryotic cells, which are
used for the expression of the nucleic acid. In one embodiment the
eukaryotic cells are mammalian cells. In another embodiment the
mammalian host cell is selected from the mammalian cells comprising
CHO cells (e.g. CHO K1 or CHO DG44), BHK cells, NS0 cells, SP2/0
cells, HEK 293 cells, HEK 293 EBNA cells, PER.C6 cells, and COS
cells. In a further embodiment the mammalian cell is selected from
the group comprising hybridoma, myeloma, and rodent cells. Myeloma
cells comprise rat myeloma cells (e.g. YB2), and mouse myeloma
cells (e.g. NS0, SP2/0). Polypeptides for use in pharmaceutical
applications are in one embodiment produced in mammalian cells such
as CHO cells, NS0 cells, Sp2/0 cells, COS cells, HEK cells, BHK
cells, PER.C6.RTM. cells, or the like. For the fermentation of the
host cell and thus for the expression of the polypeptide of
interest a cultivation medium is used. Today CHO cells are widely
used for the expression of pharmaceutical polypeptides, either at
small scale in the laboratory or at large scale in production
processes. Due to their wide distribution and use the
characteristic properties and the genetic background of CHO cells
is well known. Therefore, CHO cells are approved by regulatory
authorities for the production of therapeutic proteins for
application to human beings. In one embodiment the mammalian cell
is a CHO cell.
An "expression cassette" refers to a nucleic acid that contains the
elements necessary for expression and secretion of at least the
contained structural gene in a host cell. A nucleic acid is
likewise characterized by its sequence consisting of individual
nucleotides or by the amino acid sequence encoded by the nucleic
acid molecule.
A "gene" denotes a nucleic acid which is a segment e.g. on a
chromosome or on a plasmid which can effect the expression of a
peptide, polypeptide, or protein. Beside the coding region, i.e.
the structural gene, a gene comprises other functional elements
e.g. a signal sequence, promoter(s), introns, and/or
terminators.
A "structural gene" denotes the region of a gene without a signal
sequence, i.e. the coding region.
The term "expression" as used herein refers to transcription and/or
translation occurring within a cell. The level of transcription of
a desired product in a host cell can be determined on the basis of
the amount of corresponding mRNA that is present in the cell. For
example, mRNA transcribed from a selected nucleic acid can be
quantitated by PCR or by Northern hybridization (see Sambrook, et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press (1989)). The protein encoded by a selected nucleic
acid can be quantitated by various methods, e.g. by ELISA, by
assaying for the biological activity of the protein, or by
employing assays that are independent of such activity, such as
Western blotting or radioimmunoassay, by using antibodies that
recognize and bind to the protein (see Sambrook, et al., 1989,
supra).
"Regulatory elements" as used herein, refer to nucleotide sequences
present in cis, necessary for transcription and/or translation of
the nucleic acid sequence encoding a polypeptide of interest. The
transcriptional regulatory elements normally comprise a promoter
upstream of the nucleic acid sequence to be expressed,
transcriptional initiation and termination sites, and a
polyadenylation signal sequence. The term "transcriptional
initiation site" refers to the nucleotide in the nucleic acid
corresponding to the first nucleotide incorporated into the primary
transcript, i.e. the mRNA precursor; the transcriptional initiation
site may overlap with the promoter sequence. The term
"transcriptional termination site" refers to a nucleotide sequence
normally represented at the 3' end of a gene of interest to be
transcribed, that causes RNA polymerase to terminate transcription.
The polyadenylation signal sequence, or poly-A addition signal
provides the signal for the cleavage at a specific site at the 3'
end of eukaryotic mRNA and the post-transcriptional addition in the
nucleus of a sequence of about 100-200 adenine nucleotides (polyA
tail) to the cleaved 3' end. The polyadenylation signal sequence
may include the consensus sequence AATAAA located at about 10-30
nucleotides upstream from the site of cleavage.
A "polypeptide" is a polymer of amino acid residues joined by
peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 20 amino acid residues may be
referred to as "peptides." Polypeptides comprising two or more
amino acid chains or comprising an amino acid chain of a length of
100 amino acids or more may be referred to as "proteins". A
polypeptide or protein may also comprise non-peptidic components,
such as carbohydrate groups or metal ions. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and may vary with the type of cell.
Proteins and polypeptides are defined herein in terms of their
amino acid backbone structure; additions such as carbohydrate
groups are generally not specified, but may be present
nonetheless.
"Heterologous DNA" or "heterologous polypeptide" refers to a DNA
molecule or a polypeptide, or a population of DNA molecules or a
population of polypeptides, that do not exist naturally within a
given host cell. DNA molecules heterologous to a particular host
cell may contain DNA derived from the host cell species (i.e.
endogenous DNA) so long as that host cell derived DNA is combined
with non-host cell derived DNA (i.e. exogenous DNA). For example, a
DNA molecule containing a non-host DNA segment encoding a
polypeptide operably linked to a host DNA segment comprising a
promoter is considered to be a heterologous DNA molecule.
Conversely, a heterologous DNA molecule can comprise an endogenous
structural gene operably linked with an exogenous promoter. A
peptide or polypeptide encoded by a non-host DNA molecule is a
"heterologous" peptide or polypeptide.
The term "selectable marker" denotes a nucleic acid that allows
cells carrying this nucleic acid to be specifically selected for or
against, in the presence of a corresponding "selection agent". A
useful positive selectable marker is e.g. an antibiotic resistance
gene. The selectable marker allows a cell which is transformed
therewith to be selected for in the presence of the corresponding
selection agent; a non-transformed cell is not capable to grow or
survive under selective culture conditions, i.e. in the presence of
the selection agent. Selectable markers can be positive, negative
or bifunctional. Positive selectable markers allow the selection of
cells carrying the marker, whereas negative selectable markers
allow cells carrying the marker to be selectively eliminated.
Typically, a selectable marker will confer resistance to a drug or
compensate for a metabolic or catabolic defect in the cell.
Selectable markers useful with eukaryotic cells include, e.g., the
genes for aminoglycoside phosphotransferase (APH), such as the
hygromycin phosphotransferase (HYG), neomycin and G418 APH,
dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine
synthetase (GS), asparagine synthetase, tryptophan synthetase
(selection agent indole), histidinol dehydrogenase (selection agent
histidinol D), and genes providing resistance to puromycin,
bleomycin, phleomycin, chloramphenicol, Zeocin, and mycophenolic
acid. Further selectable markers are reported in WO 92/08796 and WO
94/28143.
The term "expression machinery" as used within the current
invention denotes the sum of the enzymes, cofactors, etc. of a
cell, which are involved in the process beginning with the
transcription of a nucleic acid or gene (i.e. also called "gene
expression machinery") to the post-translational modification of
the polypeptide encoded by the nucleic acid. The "expression
machinery" e.g. comprises the steps of transcription of DNA into
pre-mRNA, pre-mRNA splicing to mature mRNA, translation of the mRNA
into a polypeptide, and post translational modification of the
polypeptide.
The term "under conditions suitable for the expression of a
heterologous polypeptide" denotes conditions which are used for the
cultivation of a mammalian cell expressing a heterologous
polypeptide and which are known to or can easily be determined by a
person skilled in the art. It is also known to a person skilled in
the art that these conditions may vary depending on the type of
mammalian cell cultivated and type of protein expressed. In general
the mammalian cell is cultivated at a temperature, e.g. between
20.degree. C. and 40.degree. C., and for a period of time
sufficient to allow effective protein production, e.g. for 4 to 28
days, in a volume of from 0.1 liter to 10.sup.7 liter.
The term "under conditions suitable for the growth of the
non-transfected cell" denotes conditions which are generally used
for the cultivation of a non-transfected cell of the same cell
line. These conditions are known or can easily be determined by a
person skilled in the art.
The term "recovering of the heterologous polypeptide" as used
within the current application denotes precipitation, salting out,
ultrafiltration, diafiltration, lyophilization, solvent volume
reduction to obtain a concentrated solution, or chromatography.
Generally chromatographic processes are used for the separation and
purification of polypeptides. Different methods are well
established and widespread used for protein recovery and
purification, such as affinity chromatography with microbial
proteins (e.g. protein A or protein G affinity chromatography), ion
exchange chromatography (e.g. cation exchange (carboxymethyl
resins), anion exchange (amino ethyl resins) and mixed-mode
exchange), thiophilic adsorption (e.g. with beta-mercaptoethanol
and other SH ligands), hydrophobic interaction or aromatic
adsorption chromatography (e.g. with phenyl-sepharose,
aza-arenophilic resins, or m-aminophenylboronic acid), metal
chelate affinity chromatography (e.g. with Ni(II)- and
Cu(II)-affinity material), size exclusion chromatography, and
electrophoretical methods (such as gel electrophoresis, capillary
electrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75
(1998) 93-102).
The term "immunoglobulin" refers to a protein consisting of one or
more polypeptide(s) substantially encoded by immunoglobulin genes.
The recognized immunoglobulin genes include the different constant
region genes as well as the myriad immunoglobulin variable region
genes. Immunoglobulins may exist in a variety of formats,
including, for example, Fv, Fab, and F(ab)2 as well as single
chains (scFv) or diabodies (e.g. Huston, J. S., et al., Proc. Natl.
Acad. Sci. USA 85 (1988) 5879-5883; Bird, R. E., et al., Science
242 (1988) 423-426; in general, Hood, et al., Immunology, Benjamin
N.Y., 2nd edition (1984); and Hunkapiller, T. and Hood, L., Nature
323 (1986) 15-16).
An immunoglobulin in general comprises two so called light chain
polypeptides (light chain) and two so called heavy chain
polypeptides (heavy chain). Each of the heavy and light chain
polypeptides contains a variable domain (variable region)
(generally the amino terminal portion of the polypeptide chain)
comprising binding regions that are able to interact with an
antigen. Each of the heavy and light chain polypeptides comprises a
constant region (generally the carboxyl terminal portion). The
constant region of the heavy chain mediates the binding of the
antibody i) to cells bearing a Fc gamma receptor (Fc.gamma.R), such
as phagocytic cells, or ii) to cells bearing the neonatal Fc
receptor (FcRn) also known as Brambell receptor. It also mediates
the binding to some factors including factors of the classical
complement system such as component (Clq). The variable domain of
an immunoglobulin's light or heavy chain in turn comprises
different segments, i.e. four framework regions (FR) and three
hypervariable regions (CDR).
An "immunoglobulin fragment" denotes a polypeptide comprising at
least one domain of the group of domains comprising the variable
domain, the C.sub.H1 domain, the hinge-region, the C.sub.H2 domain,
the C.sub.H3 domain, the C.sub.H4 domain of a heavy chain of an
immunoglobulin or the variable domain or the C.sub.L domain of a
light chain of an immunoglobulin. Also comprised are derivatives
and variants thereof. Additionally a variable domain, in which one
or more amino acids or amino acid regions are deleted, may be
present.
An "immunoglobulin conjugate" denotes a polypeptide comprising at
least one domain of an immunoglobulin heavy or light chain
conjugated via a peptide bond to a further polypeptide. The further
polypeptide is a non-immunoglobulin peptide, such as a hormone,
growth receptor, antifusogenic peptide or the like.
The current invention reports a promoter with a nucleotide sequence
of SEQ ID NO: 02, or SEQ ID NO: 03, or SEQ ID NO: 04, or SEQ ID NO:
06
A method for the identification of a potential high producer cell
clone is the linking of the expression of a selectable marker gene
and a structural gene encoding a heterologous polypeptide via an
internal ribosome entry site (IRES). With this design the
expression of the heterologous polypeptide can be correlated with
the expression of the selectable marker. Another method is gene
amplification. Therein cells deficient of the enzyme dihydrofolate
reductase (DHFR) are transfected with a vector/plasmid which
contains a first expression cassette for the expression of the DHFR
protein and a second expression cassette for the expression of a
heterologous polypeptide. By using a culture medium depleted of
glycine, hypoxanthine and thymidine selective culture conditions
are established. For amplification a DHFR inhibitor, methotrexate
(MTX), is added (Kaufman, R. J., et al., J Mol. Biol. 159 (1982)
601-621; U.S. Pat. No. 4,656,134). Generally may be used any kind
of gene whose expression product is located/can be detected on the
cell surface as a marker for enrichment and selection of
transfectants. dLNGFR, a truncated form of the low-affinity nerve
growth factor receptor, and thus inactive for signal transduction,
which is expressed on the cell surface, and has proven to be a
highly useful marker for cell biological analysis (Philipps, K., et
al., Nat. Med. 2 (1996) 1154-1156 and Machl, A. W., et al.,
Cytometry 29 (1997) 371-374).
In order not to unnecessarily reduce the production of a
heterologous polypeptide of interest the expression of the
selectable marker, which is required for the selection of cells
producing the heterologous polypeptide, i.e. of successfully
transfected cells, should be as low as possible but nonetheless
still detectable.
It has now surprisingly been found that this need can be fulfilled
with a promoter according to the invention. By employing a promoter
according to the current invention cells can be selected which
express a heterologous polypeptide at a higher level compared to
cells selected under the same conditions and not employing a
promoter according to the current invention. It has surprisingly
been found that with a promoter according to the invention a cell
expressing a heterologous polypeptide can be isolated with reduced
expenditure. Additionally it has been found that by employing a
promoter according to the current invention cells can be selected
that express a heterologous polypeptide at a higher level compared
to cells selected by employing a full length SV40 promoter under
the same conditions and selection agent concentrations.
The term "5' shortened SV40 promoter" as used within the current
application denotes a wild-type SV40 promoter in which a defined
number of consecutive nucleotides at the 5' end of the nucleic acid
sequence have been deleted.
Thus, the current invention reports a promoter having, i.e. with,
the nucleic acid sequence of SEQ ID NO: 02. SEQ ID NO: 02 comprises
nucleotides 61 to 348 of the wild-type SV40 promoter of SEQ ID NO:
05, i.e. nucleotides 1 to 60 have been deleted. The preparation of
the promoter with SEQ ID NO: 02 is shown in Example 1.
The current invention also reports a promoter having, i.e. with,
the nucleic acid sequence of SEQ ID NO: 03. SEQ ID NO: 03
corresponds to nucleotides 130 to 348 of the wild-type SV40
promoter of SEQ ID NO: 05, i.e. nucleotides 1 to 129 have been
deleted. The preparation of the promoter with SEQ ID NO: 03 is
shown in Example 2.
The current invention finally reports a promoter having, i.e. with,
the nucleic acid sequence of SEQ ID NO: 04. SEQ ID NO: 04 is
nucleotides 177 to 348 of the wild-type SV40 promoter of SEQ ID NO:
05, i.e. nucleotides 1 to 176 have been deleted. The preparation of
the promoter with SEQ ID NO: 04 is shown in Example 3.
The current invention finally reports a promoter having, i.e. with,
the nucleic acid sequence of SEQ ID NO: 06. SEQ ID NO: 06 consists
of nucleotides 203 to 348 of the wild-type SV40 promoter of SEQ ID
NO: 05, i.e. nucleotides 1 to 202 have been deleted.
To determine the promoter strength of the promoters with a nucleic
acid sequence of SEQ ID NO: 02 to 04 and 06 expression plasmids
have been generated in which each of the different promoters is
operably linked to a nucleic acid encoding GFP (green fluorescent
protein, SEQ ID NO: 07). As can be seen from FIGS. 7 a) to c) the
5' deletion of nucleotides in the wild-type SV40 promoter nucleic
acid reduces the promoter strength. The promoter of SEQ ID NO: 02
has approximately the same strength as the full-length wild-type
SV40 promoter. The promoters of SEQ ID NO: 03 and 04 have promoter
strength of approximately 56% and approximately 19%, respectively.
Thus with the promoters according to the current invention the
expression of a nucleic acid operably linked thereto can be reduced
or limited compared to the wild-type SV40 promoter.
In simian virus 40 is the SV40 promoter preceded by two 72 bp
repeats. In one embodiment of the current invention is the first 72
bp repeat deleted and the second 72 bp repeat maintained. In one
embodiment the nucleic acid according to the invention comprises
the nucleic acid of SEQ ID NO: 14 prior to the nucleic acid of SEQ
ID NO: 04. In another embodiment the nucleic acid according to the
invention comprises the second 72 bp repeat of SEQ ID NO: 14 of the
simian virus 40 promoter. In a further embodiment in the nucleic
acid according to the invention the first 72 bp repeat of the SV40
promoter is deleted and the second 72 bp repeat of the SV40
promoter is maintained. This is useful for the expression of a
heterologous polypeptide. In this embodiment is the promoter
according to the current invention, which is only containing the
second 72 bp repeat of the wild type SV40 promoter, operably linked
to a nucleic acid encoding a selectable marker. With the reduced
promoter strength of this promoter the expression of the selectable
marker is reduced whereas the expression of the heterologous
polypeptide is maintained by using e.g. the wild type SV40 promoter
of SEQ ID NO: 05. Thus, it has been found that the combination of a
promoter according to the invention operably linked to a nucleic
acid encoding a selectable marker and of a wild-type promoter, e.g.
SV40 or CMV, operably linked to a nucleic acid encoding a
heterologous polypeptide of interest results in an improved
expression of the heterologous polypeptide compared to constructs
in which the nucleic acid encoding a selectable marker as well as
the nucleic acid encoding the heterologous polypeptide of interest
are both operably linked to a wild-type promoter.
In stable cell clones the nucleic acid encoding the selectable
marker and the nucleic acid encoding the heterologous polypeptide
as well as their corresponding promoters are integrated jointly in
the genome of said cell. As the location of the integration in the
genome is a random process a selection step is normally carried
out. In this selection step only cells are selected in which the
joint nucleic acids are incorporated in the genome is close
proximity of a transcriptionally highly active locus. Cells either
having incorporated the nucleic acid afar from such a locus or
having incorporated both nucleic acids at different loci are
eliminated by the selection step.
Another aspect of the current invention is a nucleic acid
consisting of a nucleic acid sequence of SEQ ID NO: 02, or SEQ ID
NO: 03, or SEQ ID NO: 04, or SEQ ID NO: 06, which has a promoter
strength of 90% or more, or 40% to less than 90%, or less than 40%
of the promoter strength of the wild-type SV40 promoter of SEQ ID
NO: 05 when operably linked to the nucleic acid of SEQ ID NO:
07.
In a preferred embodiment has the nucleic acid the nucleic acid
sequence of SEQ ID NO: 04 and a promoter strength of 20% or less of
the promoter strength of the wild-type SV40 promoter of SEQ ID NO:
05 when each of them individually is operably linked to the nucleic
acid of SEQ ID NO: 07.
As the nucleic acid and the promoter, respectively, according to
the invention each has reduced promoter strength, i.e. a nucleic
acid operably linked thereto is transcribed at a reduced amount or
with a reduced rate when compared to the wild-type SV40 promoter,
they are useful in multiple applications.
For example, they can be used to promote the expression of an
operably linked selection marker allowing for the selection of a
cell carrying this selection marker without requiring a large
fraction of the capacity of the cell's protein expression
machinery. Thereby the expression of an e.g. co-expressed
heterologous polypeptide is not negatively affected.
Another aspect of the current invention is a method for the
selection of a cell expressing a heterologous polypeptide
comprising the steps of a) transfecting a eukaryotic cell with a
nucleic acid comprising i) a first expression cassette comprising a
nucleic acid encoding a heterologous polypeptide, ii) a second
expression cassette comprising a first nucleic acid of SEQ ID NO:
04 and a second nucleic acid encoding a selectable marker, whereby
the first nucleic acid is operably linked to the second nucleic
acid, b) cultivating said transfected cell under conditions
suitable for growth of the non-transfected eukaryotic cell, c)
selecting a cell propagating in step b) and also i) propagating
under selection condition, or ii) expressing the selectable
marker.
Cells suitable in this method are e.g. CHO cells, BHK cells,
PER.C6.RTM. cells, HEK cells, HeLa cells, SP2/0 cells, NS0 cells,
myeloma cells, or hybridoma cells. In one embodiment the cell is a
mammalian cell, in a preferred embodiment the cell is selected from
a CHO cell, BHK cell, HEK cell, Sp2/0 cell, or a PER.C6.RTM.
cell.
The heterologous polypeptide may be any heterologous polypeptide of
interest, such as e.g. prodrugs, enzymes, enzyme fragments, enzyme
inhibitors, enzyme activators, biologically active polypeptides,
hedgehog proteins, bone morphogenetic proteins, growth factors,
erythropoietin, thrombopoietin, G-CSF, interleukins, interferons,
immunoglobulins, or antifusogenic peptides, or fragments thereof,
or conjugates thereof. In one embodiment the heterologous
polypeptide is an immunoglobulin, or an immunoglobulin fragment, or
an immunoglobulin conjugate.
In one embodiment step c) of the method is selecting a cell
propagating in step b) under selective culture conditions, i.e. in
the presence of a selection agent. In another embodiment step c) of
the method is selecting a cell propagating in step b) and
expressing the selectable marker encoded by said second nucleic
acid. In the first embodiment is the transfected cell cultivated in
the presence of a selection agent that inhibits the propagation of
cells not transfected or not sufficiently expressing the second
nucleic acid encoding the selectable marker. In the second
embodiment is the transfected cell cultivated in the absence of a
selection agent and selection is by the detection of the expression
of the selectable marker, e.g. by FACS or sight inspection.
Selection of cells can be performed in a single step or in multiple
steps. In a single/multiple step procedure the first selection can
be performed based e.g. on a threshold level of a selectable
marker, such as e.g. dLNGFR or GFP. For example, for selection by
flow cytometry (e.g. by FACS--Fluorescence Activated Cell Sorting)
a fluorescence threshold level is set and cells with a fluorescence
above this threshold level are selected. Alternatively cells within
the top 1-15% (i.e. the 15% of the cells with the most intense
detectable label), or top 1-10%, or top 1-5%, or top 5-10% of
fluorescence intensity of the sample population can be collected.
An alternative method for the selection of a cell is immunological
binding, e.g. to magnetic beads coated with Protein A or specific
immunoglobulins. The selected panel of cells may be taken as basic
population for a further selection step, e.g. by single cell
seeding, cultivation and ELISA analysis (Enzyme-linked
Immunosorbent Assay), or by limited dilution cloning, or by
expanding by cultivation for several days and a further FACS
selection, or by a further FACS selection with a higher threshold
level, which can for example be based on the fluorescence
intensities detected in a preceding FACS selection, or by an
immunoprecipitation method (see e.g. WO 2005/020924). Selecting a
cell according to the invention can in one embodiment be performed
by a method selected from flow cytometry, ELISA,
immunoprecipitation, immunoaffinity column chromatography, magnetic
bead immunoaffinity sorting, microscopy-based isolation methods, or
immunological binding. In another embodiment selecting a cell
according to the invention can be performed by a method selected
from flow cytometry, ELISA, immunoprecipitation, immunoaffinity
column chromatography, magnetic bead immunoaffinity sorting,
microscopy-based isolation methods, or immunological binding,
followed by a method selected from single cell seeding and
cultivation, limited dilution, or expanding by cultivation,
followed by a method selected from FACS, immunoprecipitation,
immunoaffinity column chromatography, magnetic bead immunoaffinity
sorting, microscopy-based isolation methods, or ELISA.
The final aspect of the current invention is a method for the
expression of a heterologous polypeptide in a cell by operably
linking a promoter according to the invention to a nucleic acid
encoding said heterologous polypeptide. This method is suitable for
the expression e.g. of large proteins with low solubilities or slow
folding kinetics. The reduction of the amount or rate of expression
of a heterologous polypeptide or of the transcription of a nucleic
acid is advisable if the heterologous polypeptide or nucleic acid
adversely affects the host cell or reduces the overall production
yield of functionable, i.e. correctly folded, heterologous
polypeptide. Therefore, one aspect of the current invention is the
expression or production of a heterologous polypeptide with reduced
fraction of not functionable, i.e. not correctly folded,
polypeptide. If the heterologous polypeptide expressed in the host
cell e.g. exceeds a certain size with respect to weight, or amino
acid number, or number of subunits, or number of secondary
modifications, it probably will be obtained after the cultivation
of the host cell in a non-functionable, i.e. non-active or not
correctly folded, form. One possibility to circumvent this problem
is to reduce the amount, i.e. the rate, of the protein expression.
As protein expression is regulated by the strength of the operably
linked promoter, the promoters according to the invention are well
suited therefore.
Therefore, the current invention comprises a method for the
expression or production of a heterologous polypeptide with reduced
fraction of not functionable polypeptide wherein the method
comprises the following steps in this order: a) transfecting a
mammalian cell with a nucleic acid comprising an expression
cassette comprising a promoter of SEQ ID NO: 02, or SEQ ID NO: 03,
or SEQ ID NO: 04, or SEQ ID NO: 06 operably linked to a nucleic
acid encoding a heterologous polypeptide, b) selecting a cell
transfected in step a), c) cultivating the selected cell under
conditions suitable for the expression of the heterologous
polypeptide, d) recovering the heterologous polypeptide from the
cell or the cultivation medium.
In one embodiment of this aspect of the current invention the
mammalian cell is a CHO cell, a BHK cell, a HEK cell, a Sp2/0 cell,
or a PER.C6.RTM. cell. In one embodiment of this method the
promoter is of SEQ ID NO: 03 or SEQ ID NO: 04. In another
embodiment has the promoter the SEQ ID NO: 04. In a further
embodiment is the nucleic acid encoding a heterologous polypeptide
encoding an immunoglobulin, or an immunoglobulin-fragment, or an
immunoglobulin-conjugate. In still another embodiment comprises the
nucleic acid a second expression cassette encoding a selectable
marker.
The following examples, sequence listing and figures are provided
to aid the understanding of the present invention, the true scope
of which is set forth in the appended claims. It is understood that
modifications can be made in the procedures set forth without
departing from the spirit of the invention.
DESCRIPTION OF THE FIGURES
FIG. 1 Plasmid map of plasmid 5500.
FIG. 2 Plasmid map of plasmid 5501.
FIG. 3 Plasmid map of plasmid 4703.
FIG. 4 Plasmid map of plasmid 4712.
FIG. 5 Plasmid map of plasmid 4713.
FIG. 6 FACS analysis of dLNGFR-expression of HEK293EBNA-cells
transfected with a) an expression cassette of SEQ ID NO: 05
operably linked to SEQ ID NO: 07, b) an expression cassette of SEQ
ID NO: 04 operably linked to SEQ ID NO: 07, c) an expression
cassette of SEQ ID NO: 06 operably linked to SEQ ID NO: 07.
FIG. 7 FACS analysis of GFP-expression of HEK293EBNA-cells
transfected with a) an expression cassette of SEQ ID NO: 05
operably linked to SEQ ID NO: 07, b) an expression cassette of SEQ
ID NO: 03 operably linked to SEQ ID NO: 07, c) an expression
cassette of SEQ ID NO: 06 operably linked to SEQ ID NO: 07.
EXAMPLE 1
Construction of Nucleic Acid of SEQ ID NO: 02
The 5' shortened SV40 promoter of SEQ ID NO: 02 was obtained via a
PCR reaction with the full length SV40 promoter as template
operably linked to a nucleic acid encoding dLNGFR (plasmid 4788).
The PCR mixture was: 1.times.PWO buffer (Roche Molecular
Biochemicals, Mannheim, Germany) supplemented with 2 mM MgSO.sub.4,
200 .mu.M dNTPs PCR Nucleotide Mix (Roche Molecular Biochemicals,
Mannheim, Germany), 1 .mu.M forward primer of SEQ ID NO: 08, 1
.mu.M reverse primer of SEQ ID NO: 13, 50 ng template-DNA of
plasmid 4788, 2.5 U PWO-DNA polymerase (PWO=Pyrococcus woesei;
Roche Molecular Biochemicals, Mannheim, Germany), ad 100 .mu.L with
doubly distilled ultrapure water. The PCR conditions were: 1 min at
94.degree. C., 1 cycle; 0.5 min at 94.degree. C., 25 cycles; 0.5
min at 55.degree. C., 25 cycles; 1 min at 72.degree. C., 25 cycles;
5 min at 72.degree. C., 1 cycle.
EXAMPLE 2
Construction of Nucleic Acid of SEQ ID NO: 03
The 5' shortened SV40 promoter variant of SEQ ID NO: 03 was
obtained via a PCR reaction with the full length SV40 promoter as
template operably linked to a nucleic acid encoding dLNGFR from
plasmid 4788. The PCR mixture was: 1.times.PWO buffer supplemented
with 2 mM MgSO.sub.4, 200 .mu.M dNTPs PCR Nucleotide Mix, 1 .mu.M
forward primer of SEQ ID NO: 09, 1 .mu.M reverse primer of SEQ ID
NO: 13, 50 ng template-DNA of plasmid 4788, 2.5 U PWO-DNA
polymerase, ad 100 .mu.L with doubly distilled ultrapure water. The
PCR conditions were: 1 min at 94.degree. C., 1 cycle; 0.5 min at
94.degree. C., 25 cycles; 0.5 min at 55.degree. C., 25 cycles; 1
min at 72.degree. C., 25 cycles; 5 min at 72.degree. C., 1
cycle.
EXAMPLE 3
Construction of Nucleic Acid of SEQ ID NO: 04
The 5' shortened SV40 promoter variant of SEQ ID NO: 04 was
obtained via a PCR reaction with the full length SV40 promoter as
template operably linked to a nucleic acid encoding dLNGFR (plasmid
4788). The PCR mixture was: 1.times.PWO buffer supplemented with 2
mM MgSO.sub.4, 200 .mu.M dNTPs PCR Nucleotide Mix, 1 .mu.M forward
primer of SEQ ID NO: 10, 1 .mu.M reverse primer of SEQ ID NO: 13,
50 ng template-DNA of plasmid 4788, 2.5 U PWO-DNA polymerase, ad
100 .mu.L, with doubly distilled ultrapure water. The PCR
conditions were: 1 min at 94.degree. C., 1 cycle; 0.5 min at
94.degree. C., 25 cycles; 0.5 min at 55.degree. C., 25 cycles; 1
min at 72.degree. C., 25 cycles; 5 min at 72.degree. C., 1
cycle.
EXAMPLE 4
Construction of Further Promoters
Further 5' shortened SV40 promoter variants were produced via a PCR
reaction with the full length SV40 promoter as template operably
linked to a nucleic acid encoding dLNGFR. The PCR mixture was:
1.times.PWO buffer supplemented with 2 mM MgSO.sub.4, 200 .mu.M
dNTPs PCR Nucleotide Mix, 1 .mu.M forward primer of SEQ ID NO: 11
(yielding SEQ ID NO: 06) or SEQ ID NO: 12, 1 .mu.M reverse primer
of SEQ ID NO: 13, 50 ng template-DNA of Plasmid 4788, 2.5 U PWO-DNA
polymerase, ad 100 .mu.L with bidistilled ultrapure water. The PCR
conditions were: 1 min at 94.degree. C., 1 cycle; 0.5 min at
94.degree. C., 25 cycles; 0.5 min at 55.degree. C., 25 cycles; 1
min at 72.degree. C., 25 cycles; 5 min at 72.degree. C., 1
cycle.
EXAMPLE 5
Expression of dLNGFR Operably Linked to SEQ ID NO: 02, 03, 04, and
06
The primer with which the nucleic acids of SEQ ID NO: 02, 03, 04,
and 06 were obtained contained restriction sites of the restriction
endonucleases SalI and EcoRI. Using these restriction
sites/restriction endonucleases these nucleic acids operably linked
to a nucleic acid encoding dLNGFR (for LNGFR (low affinity nerve
growth factor) see e.g. Philipps, K., et al., Nat. Med. 2 (1996)
1154-1156; or Machl, A. W., et al., Cytometry 29 (1997) 371-374)
have been ligated into the plasmid 4736-pUC-DHFR, which has been
linearized using the restriction sites SalI and PvulI. The
resulting plasmids are:
5500-pUC-DHFR_dLNGFR_wildtypeSV40 (plasmid map in FIG. 1),
5501-pUC-DHFR_dLNGFR_Shortening.sub.--2 (plasmid map in FIG.
2),
5502-pUC-DHFR_dLNGFR_Shortening.sub.--3,
5503-pUC-DHFR_dLNGFR_Shortening.sub.--4,
5504-pUC-DHFR_dLNGFR_Shortening.sub.--6.
HEK 293 EBNA cells have been transfected with these plasmids and
the encoded polypeptide was transiently expressed. After 48 h the
expression of dLNGFR has been verified via FACS. For the
determination of the promoter strength (expression strength) of the
different promoters the expressed surface marker dLNGFR was
fluorescence marked via an anti-dLNGFR antibody.
For each determination approximately 0.5.times.10.sup.6 to
1.0.times.10.sup.6 cells have been detached by the addition of 1 ml
Accutase.RTM. per 6 wells (GIBCO Invitrogen, Karlsruhe, Germany).
The detached cells were transferred in a vial and washed once with
RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum.
Afterwards the cells were precipitated by centrifugation (1,500
rpm, 5 min.) and the supernatant was discarded. All following steps
were performed at 0 to 2.degree. C. on or in an ice bath. The cell
pellet was resuspended in 100 .mu.l of a solution containing the
anti-dLNGFR antibody at 30 .mu.g/ml. After an incubation period of
30 minutes the samples were diluted by the addition of 2 ml of
ice-cold RPMI 1640 medium with subsequent precipitation by
centrifugation. The pellet was resuspended in 100 .mu.L of a
secondary antibody solution, a goat anti-mouse-IgG antibody
conjugated to Phycoerythrin (Caltag Laboratories, Burlingame,
Calif., USA), at a concentration of 20 .mu.g/ml. The sample was
incubated in the dark for 30 min. on ice. After a washing and
centrifugation step the sample was resuspended in 500 .mu.l medium
and stored in the dark on ice until the measurement. The FACS
analysis was evaluated using the FACSCalibur software (Cell Quest
Pro). The results are shown in FIG. 6.
Results of the FACS analysis:
5500-pUC-DHFR_dLNGFR_wildtypeSV40 (FIG. 6a)):
TABLE-US-00001 Marker Left, Right Events % Gated % Total Mean
Median All 1, 9910 8067 100.00 80.67 567.61 128.64 M1 1, 40 2134
26.45 21.34 21.84 21.29 M2 40, 9910 5956 73.83 59.56 761.11
345.99
5501-pUC-DHFR_dLNGFR_Shortening.sub.--2:
TABLE-US-00002 Marker Left, Right Events % Gated % Total Mean
Median All 1, 9910 7377 100.00 73.77 564.33 168.49 M1 1, 40 1365
18.50 13.65 24.69 25.48 M2 40, 9910 6027 81.70 60.27 685.24
291.64
5502-pUC-DHFR_dLNGFR_Shortening.sub.--3:
TABLE-US-00003 Marker Left, Right Events % Gated % Total Mean
Median All 1, 9910 7643 100.00 76.43 582.85 129.80 M1 1, 40 1959
25.63 19.59 22.68 22.88 M2 40, 9910 5708 74.68 57.08 772.82
339.82
5503-pUC-DHFR_dLNGFR_Shortening.sub.--4 (FIG. 6b)):
TABLE-US-00004 Marker Left, Right Events % Gated % Total Mean
Median All 1, 9910 7440 100.00 74.40 436.61 69.78 M1 1, 40 2603
34.99 26.03 20.87 19.99 M2 40, 9910 4852 65.22 48.52 658.42
250.29
5504-pUC-DHFR_dLNGFR_Shortening.sub.--6 (FIG. 6c)):
TABLE-US-00005 Marker Left, Right Events % Gated % Total Mean
Median All 1, 9910 8404 100.00 84.04 31.67 11.65 M1 1, 40 6685
79.55 66.85 12.05 9.14 M2 40, 9910 1732 20.61 17.32 107.45
74.32
It can be seen that the mean fluorescence intensity of the labeled
dLNGFR expressed from plasmid 5504 shows a significant reduction of
expression with about 85% reduction.
EXAMPLE 6
Expression of GFP Operably Linked to SEQ ID NO: 02, 03, 04, and
06
The primer with which the nucleic acids of SEQ ID NO: 02, 03, 04,
and 06 were obtained contained restriction sites of the restriction
endonucleases SalI and EcoRI. Using these restriction
sites/restriction endonucleases these nucleic acids operably linked
to a nucleic acid encoding GFP (SEQ ID NO: 07) have been ligated
into the plasmid 4703-pUC-OriP (FIG. 3), which has been linearized
using the restriction sites SalI and PvulI. The resulting plasmids
were:
4712-pUC-Hyg_GFP_wildtypeSV40 (plasmid map in FIG. 4),
4713-pUC-Hyg_GFP_Shortening.sub.--2 (plasmid map in FIG. 5),
4714-pUC-Hyg_GFP_Shortening.sub.--3,
4715-pUC-Hyg_GFP_Shortening.sub.--4,
4716-pUC-Hyg_GFP_Shortening.sub.--6.
For each determination approximately 5.times.10.sup.5 to
1.times.10.sup.6 cells have been detached by the addition of 1 ml
Accutase.RTM. per 6 wells (GIBCO Invitrogen, Karlsruhe, Germany).
The detached cells were transferred in a vial and resuspended in 3
ml RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum.
Afterwards the cells were precipitated by centrifugation (1,500
rpm, 5 min.) and the supernatant was discarded. The cell pellet was
resuspended in 500 .mu.l medium. For the differentiation of living
and dead cells 1 .mu.l propidium iodide was added. The cells were
resuspended shortly prior to the FACS measurement. The FACS
analysis was evaluated using the FACSCalibur software (Cell Quest
Pro). The results are shown in FIG. 7.
Results of the FACS Analysis:
4712-pUC-Hyg_GFP_wildtypeSV40 (FIG. 7a)):
TABLE-US-00006 Marker Left, Right Events % Gated % Total Mean
Median All 1, 9910 8390 100.00 33.90 385.26 17.15 M1 1, 16 4162
49.61 41.62 5.92 4.91 M2 16, 9910 4240 50.54 42.40 756.58
302.32
4713-pUC-Hyg_GFP_Shortening.sub.--2:
TABLE-US-00007 Marker Left, Right Events % Gated % Total Mean
Median All 1, 9910 8576 100.00 85.76 514.24 45.73 M1 1, 16 3635
42.39 36.35 5.72 4.61 M2 16, 9910 4948 57.70 49.48 887.12
392.42
4714-pUC-Hyg_GFP_Shortening.sub.--3 (FIG. 7b):
TABLE-US-00008 Marker Left, Right Events % Gated % Total Mean
Median All 1, 9910 8538 100.00 85.38 215.22 15.96 M1 1, 16 4289
50.23 42.89 5.44 4.26 M2 16, 9910 4258 49.87 42.58 426.11
203.51
4715-pUC-Hyg_GFP_Shortening.sub.--4:
TABLE-US-00009 Marker Left, Right Events % Gated % Total Mean
Median All 1, 9910 8601 100.00 86.01 53.76 7.37 M1 1, 16 5606 65.18
56.06 5.59 4.41 M2 16, 9910 3012 35.02 30.12 143.20 73.65
4716-pUC-Hyg_GFP_Shortening.sub.--6 (FIG. 7c)):
TABLE-US-00010 Marker Left, Right Events % Gated % Total Mean
Median All 1, 9910 7622 100.00 76.22 4.09 3.52 M1 1, 16 7614 99.90
76.14 4.07 3.52 M2 16, 9910 8 0.10 0.08 22.46 18.85
It can be seen that the mean fluorescence intensity of the GFP
expressed from plasmid 4714 or plasmid 4715 shows a significant
reduction of expression with about 50% and 75% reduction,
respectively. With plasmid 4716 no detectable expression of GFP was
found.
SEQUENCE LISTINGS
1
1415243DNASimian virus 40 1gcctcggcct ctgcataaat aaaaaaaatt
agtcagccat ggggcggaga atgggcggaa 60ctgggcggag ttaggggcgg gatgggcgga
gttaggggcg ggactatggt tgctgactaa 120ttgagatgca tgctttgcat
acttctgcct gctggggagc ctggggactt tccacacctg 180gttgctgact
aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
240tttccacacc ctaactgaca cacattccac agctggttct ttccgcctca
gaaggtacct 300aaccaagttc ctctttcaga ggttatttca ggccatggtg
ctgcgccggc tgtcacgcca 360ggcctccgtt aaggttcgta ggtcatggac
tgaaagtaaa aaaacagctc aacgcctttt 420tgtgtttgtt ttagagcttt
tgctgcaatt ttgtgaaggg gaagatactg ttgacgggaa 480acgcaaaaaa
ccagaaaggt taactgaaaa accagaaagt taactggtaa gtttagtctt
540tttgtctttt atttcaggtc catgggtgct gctttaacac tgttggggga
cctaattgct 600actgtgtctg aagctgctgc tgctactgga ttttcagtag
ctgaaattgc tgctggagag 660gccgctgctg caattgaagt gcaacttgca
tctgttgcta ctgttgaagg cctaacaacc 720tctgaggcaa ttgctgctat
aggcctcact ccacaggcct atgctgtgat atctggggct 780cctgctgcta
tagctggatt tgcagcttta ctgcaaactg tgactggtgt gagcgctgtt
840gctcaagtgg ggtatagatt ttttagtgac tgggatcaca aagtttctac
tgttggttta 900tatcaacaac caggaatggc tgtagatttg tataggccag
atgattacta tgatatttta 960tttcctggag tacaaacctt tgttcacagt
gttcagtatc ttgaccccag acattggggt 1020ccaacacttt ttaatgccat
ttctcaagct ttttggcgtg taatacaaaa tgacattcct 1080aggctcacct
cacaggagct tgaaagaaga acccaaagat atttaaggga cagtttggca
1140aggtttttag aggaaactac ttggacagta attaatgctc ctgttaattg
gtataactct 1200ttacaagatt actactctac tttgtctccc attaggccta
caatggtgag acaagtagcc 1260aacagggaag ggttgcaaat atcatttggg
cacacctatg ataatattga tgaagcagac 1320agtattcagc aagtaactga
gaggtgggaa gctcaaagcc aaagtcctaa tgtgcagtca 1380ggtgaattta
ttgaaaaatt tgaggctcct ggtggtgcaa atcaaagaac tgctcctcag
1440tggatgttgc ctttacttct aggcctgtac ggaagtgtta cttctgctct
aaaagcttat 1500gaagatggcc ccaacaaaaa gaaaaggaag ttgtccaggg
gcagctccca aaaaaccaaa 1560ggaaccagtg caagtgccaa agctcgtcat
aaaaggagga atagaagttc taggagttaa 1620aactggagta gacagcttca
ctgaggtgga gtgcttttta aatcctcaaa tgggcaatcc 1680tgatgaacat
caaaaaggct taagtaaaag cttagcagct gaaaaacagt ttacagatga
1740ctctccagac aaagaacaac tgccttgcta cagtgtggct agaattcctt
tgcctaattt 1800aaatgaggac ttaacctgtg gaaatatttt gatgtgggaa
gctgttactg ttaaaactga 1860ggttattggg gtaactgcta tgttaaactt
gcattcaggg acacaaaaaa ctcatgaaaa 1920tggtgctgga aaacccattc
aagggtcaaa ttttcatttt tttgctgttg gtggggaacc 1980tttggagctg
cagggtgtgt tagcaaacta caggaccaaa tatcctgctc aaactgtaac
2040cccaaaaaat gctacagttg acagtcagca gatgaacact gaccacaagg
ctgttttgga 2100taaggataat gcttatccag tggagtgctg ggttcctgat
ccaagtaaaa atgaaaacac 2160tagatatttt ggaacctaca caggtgggga
aaatgtgcct cctgttttgc acattactaa 2220cacagcaacc acagtgcttc
ttgatgagca gggtgttggg cccttgtgca aagctgacag 2280cttgtatgtt
tctgctgttg acatttgtgg gctgtttacc aacacttctg gaacacagca
2340gtggaaggga cttcccagat attttaaaat tacccttaga aagcggtctg
tgaaaaaccc 2400ctacccaatt tcctttttgt taagtgacct aattaacagg
aggacacaga gggtggatgg 2460gcagcctatg attggaatgt cctctcaagt
agaggaggtt agggtttatg aggacacaga 2520ggagcttcct ggggatccag
acatgataag atacattgat gagtttggac aaaccacaac 2580tagaatgcag
tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
2640aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
ttatgtttca 2700ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
aacctctaca aatgtggtat 2760ggctgattat gatcatgaac agactgtgag
gactgagggg cctgaaatga gccttgggac 2820tgtgaatcaa tgcctgtttc
atgccctgag tcttccatgt tcttctcccc accatcttca 2880tttttatcag
cattttcctg gctgtcttca tcatcatcat cactgtttct tagccaatct
2940aaaactccaa ttcccatagc cacattaaac ttcatttttt gatacactga
caaactaaac 3000tctttgtcca atctctcttt ccactccaca attctgctct
gaatactttg agcaaactca 3060gccacaggtc tgtaccaaat taacataaga
agcaaagcaa tgccactttg aattattctc 3120ttttctaaca aaaactcact
gcgttccagg caatgcttta aataatcttt gggcctaaaa 3180tctatttgtt
ttacaaatct ggcctgcagt gttttaggca cactgtactc attcatggtg
3240actattccag ggggaaatat ttgagttctt ttatttaggt gtttcttttc
taagtttacc 3300ttaacactgc catccaaata atcccttaaa ttgtccaggt
tattaattcc ctgacctgaa 3360ggcaaatctc tggactcccc tccagtgccc
tttacatcct caaaaactac taaaaactgg 3420tcaatagcta ctcctagctc
aaagttcagc ctgtccaagg gcaaattaac atttaaagct 3480ttccccccac
ataattcaag caaagcagct gctaatgtag ttttaccact atcaattggt
3540cctttaaaca gccagtatct ttttttagga atgttgtaca ccatgcattt
taaaaagtca 3600tacaccactg aatccatttt gggcaacaaa cagtgtagcc
aagcaactcc agccatccat 3660tcttctatgt cagcagagcc tgtagaacca
aacattatat ccatcctatc caaaagatca 3720ttaaatctgt ttgttaacat
ttgttctcta gttaattgta ggctatcaac ccgcttttta 3780gctaaaacag
tatcaacagc ctgttggcat atggtttttt ggtttttgct gtcagcaaat
3840atagcagcat ttgcataatg cttttcatgg tacttatagt ggctgggctg
ttctttttta 3900atacatttta aacacatttc aaaactgtac tgaaattcca
agtacatccc aagcaataac 3960aacacatcat cacattttgt ttccattgca
tactctgtta caagcttcca ggacacttgt 4020ttagtttcct ctgcttcttc
tggattaaaa tcatgctcct ttaacccacc tggcaaactt 4080tcctcaataa
cagaaaatgg atctctagtc aaggcactat acatcaaata ttccttatta
4140acccctttac aaattaaaaa gctaaaggta cacaattttt gagcatagtt
attaatagca 4200gacactctat gcctgtgtgg agtaagaaaa aacagtatgt
tatgattata actgttatgc 4260ctacttataa aggttacaga atatttttcc
ataattttct tgtatagcag tgcagctttt 4320tcctttgtgg tgtaaatagc
aaagcaagca agagttctat tactaaacac agcatgactc 4380aaaaaactta
gcaattctga aggaaagtcc ttggggtctt ctacctttct cttctttttt
4440ggaggagtag aatgttgaga gtcagcagta gcctcatcat cactagatgg
catttcttct 4500gagcaaaaca ggttttcctc attaaaggca ttccaccact
gctcccattc atcagttcca 4560taggttggaa tctaaaatac acaaacaatt
agaatcagta gtttaacaca ttatacactt 4620aaaaatttta tatttacctt
agagctttaa atctctgtag gtagtttgtc caattatgtc 4680acaccacaga
agtaaggttc cttcacaaag atcaagtcca aaccacattc taaagcaatc
4740gaagcagtag caatcaaccc acacaagtgg atctttcctg tataattttc
tattttcatg 4800cttcatcctc agtaagcaca gcaagcatat gcagttagca
gacattttct ttgcacactc 4860aggccattgt ttgcagtaca ttgcatcaac
accaggattt aaggaagaag caaatacctc 4920agttgcatcc cagaagcctc
caaagtcagg ttgatgagca tattttactc catcttccat 4980tttcttgtac
agagtattca ttttcttcat tttttcttca tctcctcctt tatcaggatg
5040aaactccttg cattttttta aatatgcctt tctcatcaga ggaatattcc
cccaggcact 5100cctttcaaga cctagaaggt ccattagctg caaagattcc
tctctgttta aaactttatc 5160catctttgca aagctttttg caaaagccta
ggcctccaaa aaagcctcct cactacttct 5220ggaatagctc agaggccgag gcg
52432288DNAArtificialpromoter 1 2agtcagcaac caggtgtgga aagtccccag
gctccccagc aggcagaagt atgcaaagca 60tgcatctcaa ttagtcagca accatagtcc
cgcccctaac tccgcccatc ccgcccctaa 120ctccgcccag ttccgcccat
tctccgcccc atggctgact aatttttttt atttatgcag 180aggccgaggc
cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
240gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcg
2883219DNAArtificialpromoter 2 3attagtcagc aaccatagtc ccgcccctaa
ctccgcccat cccgccccta actccgccca 60gttccgccca ttctccgccc catggctgac
taattttttt tatttatgca gaggccgagg 120ccgcctctgc ctctgagcta
ttccagaagt agtgaggagg cttttttgga ggcctaggct 180tttgcaaaaa
gctcccggga gcttgtatat ccattttcg 2194172DNAArtificialpromoter 3
4ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
60gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
120ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cg
1725348DNASimian virus 40 5ggtgtggaaa gtccccaggc tccccagcag
gcagaagtat gcaaagcatg catctcaatt 60agtcagcaac caggtgtgga aagtccccag
gctccccagc aggcagaagt atgcaaagca 120tgcatctcaa ttagtcagca
accatagtcc cgcccctaac tccgcccatc ccgcccctaa 180ctccgcccag
ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
240aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
ttttttggag 300gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcg
3486146DNAArtificialpromoter 4 6tccgccccat ggctgactaa ttttttttat
ttatgcagag gccgaggccg cctctgcctc 60tgagctattc cagaagtagt gaggaggctt
ttttggaggc ctaggctttt gcaaaaagct 120cccgggagct tgtatatcca ttttcg
1467717DNAAequorea victoria 7atgagtaaag gagaagaact tttcactgga
gttgtcccaa ttcttgttga attagatggt 60gatgttaatg ggcacaaatt ctctgtcagt
ggagagggtg aaggtgatgc aacatacgga 120aaacttaccc ttaaatttat
ttgcactact ggaaagctac ctgttccatg gccaacactt 180gtcactactt
tctcttatgg tgttcaatgc ttttcaagat acccagatca tatgaaacag
240catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac
tatattttac 300aaagatgacg ggaactacaa atcacgtgct gaagtcaagt
ttgaaggtga taccctcgtt 360aatagaattg agttaaaagg tattgatttt
aaagaagatg gaaacattct tggacacaaa 420atggaataca actataactc
acacaatgta tacatcatgg cagacaaaca aaagaatgga 480atcaaagtta
acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac
540cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga
caaccattac 600ctgtccacac aatctgccct ttccaaagat cccaacgaaa
agagagatca catgatcctt 660cttgagtttg taacagctgc tgggattaca
catggcatgg atgaactata caaataa 717825DNAArtificialfoward primer 1
8ttagggtgtg gaaagtcccc aggct 25929DNAArtificialfoward primer 2
9aattagtcag caaccaggtg tggaaagtc 291025DNAArtificialfoward primer 3
10attagtcagc aaccatagtc ccgcc 251123DNAArtificialfoward primer 4
11ctaactccgc ccagttccgc cca 231224DNAArtificialforward primer 5
12ccgccccatg gctgactaat tttt 241322DNAArtificialreverse primer
13ggagcttgta tatccatttt cg 221472DNASimian virus 40 14tggttgctga
ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg 60actttccaca
cc 72
* * * * *